Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2015 Identification of Candidate Genes for Craniosynostosis Karen Rymer Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Diseases Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/3782 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Identification of Candidate Genes for Craniosynostosis Karen Suzanne Rymer VIRGINIA COMMONWEALTH UNIVERSITY DEPARTMENT OF HUMAN AND MOLECULAR GENETICS 2015 IDENTIFICATION OF CANDIDATE GENES FOR CRANIOSYNOSTOSIS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University By Karen Suzanne Rymer Bachelor of Science, Mathematics, Liberty University, 2013 Bachelor of Science, Biology: Premed, Liberty University, 2013 Director: RITA SHIANG, Ph.D. ASSOCIATE PROFESSOR DEPARTMENT OF HUMAN AND MOLECULAR GENETICS Virginia Commonwealth University Richmond, VA April, 2015 i Acknowledgement I would first like to thank Dr. Rita Shiang, who has guided me through this (seemingly endless) journey towards my master’s degree. I could not have come this far without her advice, constructive criticism, and extreme patience. Dr. Shiang has made my days in lab educational, yet very enjoyable. I would also like to thank my committee members for the part they all played to make this project a reality. Dr. Jennifer Rhodes made this project possible through her work in the Center for Craniofacial Care at Virginia Commonwealth University. Dr. Jim Lister’s expertise in the zebrafish model system has provided endless help for my animal model testing. Dr. Todd Webb was able to handle all of my bioinformatics and coding questions with ease. I cannot forget my lab members Anting, Laura, and Danielle. I could not have survived early mornings or late nights without the constant supply of caffeine and food that was provided (of course no food was eaten in lab). Last, but certainly not least, I would like to thank my husband, Alex, and my mom and dad for always being there for me. They have supported me through my most stressful times, as well as celebrated with me in my triumphs. No matter what happens, I know I have the love and support of my family. ii Table of Contents Table of Contents --------------------------------------------------------------------------------------------- iii List of Figures, Tables and Appendices -------------------------------------------------------------------- v List of Abbreviations ---------------------------------------------------------------------------------------- vi Abstract ------------------------------------------------------------------------------------------------------- viii Chapter 1: Introduction --------------------------------------------------------------------------------------- 1 Cranial Bone Development ------------------------------------------------------------------------------- 1 Cranial Sutures ---------------------------------------------------------------------------------------------- 4 Syndromic Craniosynostosis------------------------------------------------------------------------------ 6 Nonsyndromic Craniosynostosis ------------------------------------------------------------------------- 7 Treatment Options ---------------------------------------------------------------------------------------- 10 Genetics of Syndromic Craniosynostosis -------------------------------------------------------------- 10 FGFR Genes -------------------------------------------------------------------------------------------- 10 TWIST1-------------------------------------------------------------------------------------------------- 15 MSX2 ---------------------------------------------------------------------------------------------------- 15 Genetics of Nonsyndromic Craniosynostosis --------------------------------------------------------- 16 FGFR – Coronal Synostosis -------------------------------------------------------------------------- 16 FREM1 – Metopic Synostosis ------------------------------------------------------------------------ 16 Zebrafish as a Model Organism ------------------------------------------------------------------------- 17 Whole Exome Sequencing ------------------------------------------------------------------------------- 21 Aims --------------------------------------------------------------------------------------------------------- 21 Chapter 2: Materials and Methods ------------------------------------------------------------------------- 23 Patients ------------------------------------------------------------------------------------------------------ 23 Whole Exome Sequencing ------------------------------------------------------------------------------- 26 Mendelian Error Analysis -------------------------------------------------------------------------------- 26 Filtering of Variants --------------------------------------------------------------------------------------- 29 Quality Control of Variant Calls ------------------------------------------------------------------------ 32 PCR Primer Design --------------------------------------------------------------------------------------- 34 Sanger Sequencing ---------------------------------------------------------------------------------------- 35 In Silico Functional Prediction of Candidate Genes-------------------------------------------------- 35 CRISPR/Cas9 Clone Generation ------------------------------------------------------------------------ 35 Generation of BAMBI Overexpression Clone -------------------------------------------------------- 37 Zebrafish Culture and Maintenance -------------------------------------------------------------------- 38 Zebrafish Injection ---------------------------------------------------------------------------------------- 38 iii Staining ----------------------------------------------------------------------------------------------------- 39 Genotyping ------------------------------------------------------------------------------------------------- 40 Chapter 3: Results -------------------------------------------------------------------------------------------- 41 Whole Exome Sequencing Analysis -------------------------------------------------------------------- 41 Mendelian Error Analysis -------------------------------------------------------------------------------- 44 KGGSeq and IGV Filtering ------------------------------------------------------------------------------ 45 Sanger Sequencing ---------------------------------------------------------------------------------------- 49 CS-64 Candidate Gene Analysis ------------------------------------------------------------------------ 49 Functional Analysis of CS-64 Candidate Genes ------------------------------------------------------ 50 BAMBIa Cloning ----------------------------------------------------------------------------------------- 53 Chapter 4: Discussion ---------------------------------------------------------------------------------------- 54 Mendelian Error Analysis -------------------------------------------------------------------------------- 54 Confirmation with Sanger Sequencing ----------------------------------------------------------------- 55 FGFR3 Mutations CS-39 --------------------------------------------------------------------------------- 55 FGFR2 Mutation CS-58 Family------------------------------------------------------------------------- 57 FREM1 Mutations in CS-2 Family --------------------------------------------------------------------- 58 CS-14 Twin Pair ------------------------------------------------------------------------------------------- 59 CS-64 Family Candidate Genes ------------------------------------------------------------------------- 60 ITGAV ------------------------------------------------------------------------------------------------------ 60 SLC30A9 --------------------------------------------------------------------------------------------------- 64 BAMBI ----------------------------------------------------------------------------------------------------- 65 Five Week Staining Results ------------------------------------------------------------------------------ 67 BAMBI Overexpression ---------------------------------------------------------------------------------- 67 Future Studies ---------------------------------------------------------------------------------------------- 68 References ----------------------------------------------------------------------------------------------------- 70 Appendix ------------------------------------------------------------------------------------------------------ 75 iv List of Figures, Tables and Appendices Figure 1 -------------------------------------------------------------------------------------------------------- 3 Figure 2 -------------------------------------------------------------------------------------------------------- 4 Figure 3 -------------------------------------------------------------------------------------------------------- 9 Figure 4 ------------------------------------------------------------------------------------------------------- 14 Figure 5 -------------------------------------------------------------------------------------------------------